Renewable transportation fuels are of significant scientific, economic, environmental, and geopolitical interest due to the inherently limited supply of petroleum. Among renewable transportation fuel alternatives, large-scale generation of ethanol from lignocellulosic starting material has several advantages including a ready supply of feedstock, potential to reduce greenhouse gas emissions (e.g., depending on cultivation, harvesting, and processing methods), potential for job creation particularly in rural settings, current and projected availability of flex-fuel and dedicated ethanol-fueled vehicle technology, and distribution systems already amenable to volatile liquid fuels. However, current methods for lignocellulosic ethanol production have unfavorable chemical and/or energy requirements and therefore unacceptable cost of production, largely due to the recalcitrance of lignocellulosic feedstock to saccharification and hydrolysis in comparison to starch-rich feedstock such as milled corn kernels (See e.g., Sun et al. (2002) Bioresource Technol. 83:1-11; Hahn-Hagerdal et al. (2006) Trends Biotechnol. 24:549-556; Sanchez et al. (2007) Bioresource Technol. 99:5270-5295; each herein incorporated by reference in their entireties).
Biochemically, the major impediment to the economical use of lignocellulosic feedstock is the presence of hemicelluloses and lignins surrounding and/or cross-linking cellulose. In order for cellulase enzymes to efficiently access and degrade cellulose during the fermentation step, these hemicelluloses and lignins must have previously been at least partially degraded. For this reason, pretreatment of lignocellulosic feedstock is currently considered an economically unfortunate necessity.
Through pretreatment, feedstock is modified chemically, morphologically, and/or physically. Pretreatment methods standard in the art include exposure of lignocellulosic feedstock to high temperature and/or pressure (as with steam pretreatment or hydrothermolysis), acids or bases, or a combination of such methods (See e.g., Galbe et al. (2007) Adv. Biochem. Engin./Biotechnol. 108:41-65; Chandra et at (2007) Adv. Biochem. Engin./Biotechnol. 108:67-93; each herein incorporated by reference in their entireties). However, each of these pretreatment approaches has drawbacks. Dilute acid pretreatment (generally at high temperature, e.g. 140-200° C.) hydrolyzes hemicelluloses yielding a significant proportion of monomer sugars, but acid-hydrolyzed materials are generally difficult to ferment due to the generation of compounds that are toxic to microbes used for fermentation (See e.g., Galbe and Zacchi (2007) Adv. Biochem. Engin./Biotechnol. 108:41-65; Chandra et at (2007) Adv. Biochem. Engin./Biotechnol. 108:67-93; each herein incorporated by reference in their entireties). Alkaline pretreatment (also generally conducted at high temperature) causes at least partial delignification and solubilization of hemicelluloses as well as greater accessibility of the crystalline cellulose component of the cell wall; however, alkaline pretreatment is not suitable for all lignocellulosic feedstock types (See e.g., Galbe et al. (2007) Adv. Biochem. Engin./Biotechnol. 108:41-65; herein incorporated by reference in its entirety). Furthermore, a washing or pH adjustment step may be required for acid- or alkaline-pretreated materials to facilitate compatibility with downstream fermentation processes intolerant of low or high pH. Steam pretreatment and combinations of steam and pH treatments such as ammonia fiber explosion (AFEX) are technologies closest to commercial production, but again are not suitable for all feedstock types and have high energetic demands (See e.g., Galbe and Zacchi (2007) Adv. Biochem. Engin./Biotechnol. 108:41-65; herein incorporated by reference in its entirety). Hydrothermolysis treatment requires lower initial energy investment than steam pretreatment, but results in the need for more energy-demanding downstream processes (See e.g., Galbe and Zacchi (2007) Adv. Biochem. Engin./Biotechnol. 108:41-65; herein incorporated by reference in its entirety). Wet oxidation pretreatment (infusion of biomass with water and air or oxygen at 120° C.) is only compatible with low-lignin feedstock and renders unrecoverable any lignin that is present; this is considered detrimental from a process standpoint, as this lignin might otherwise be used as solid fuel within the biorefinery (See e.g., Galbe and Zacchi (2007) Adv. Biochem. Engin./Biotechnol. 108:41-65; herein incorporated by reference in its entirety). A further consideration is the ability to utilize residual material from biofuel production for other purposes, such as agricultural feed additives. Such secondary uses would offer economic benefit by lowering the cost of agricultural food and feed while simultaneously preventing the cost of biofuel residue disposal. However, this is generally impossible for existing technologies that render residual material unfit for consumption due to the presence of solvents, acids, bases, or by resulting in residuals that are of poor or even anti-nutritive value.